Circulating exosome long non-coding RNAs are associated with atrial structural remodeling by increasing systemic inflammation in atrial fibrillation patients

Abstract Background Atrial fibrillation (AF) is the most common cardiac arrhythmia with severe clinical sequelae, but its genetic characteristic implicated in pathogenesis has not been completely clarified. Accumulating evidence has indicated that circulating exosomes and their carried cargoes, such as long non-coding RNAs (lncRNAs), involve in the progress of multiple cardiovascular diseases. However, their potential role as clinical biomarkers in AF diagnosis and prognosis remains unknown. Methods Herein, we conducted the sequence and bioinformatic analysis of circulating exosomes harvested from AF and sinus rhythm patients. Results A total of 53 differentially expressed lncRNAs were identified, and a total of 6 significantly changed lncRNAs (fold change > 2.0), including NR0046235, NR003045, NONHSAT167247.1, NONHSAT202361.1, NONHSAT205820.1 and NONHSAT200958.1, were verified by qRT-PCR in 215 participants. Moreover, these circulating exosome lncRNA levels were different between paroxysmal and persistent AF patients, which were dramatically associated with abnormal hemodynamics and atrial diameter. Furthermore, we observed that the area under ROC curve (AUC) of six lncRNAs combination for diagnosis of persistent AF was 80.34%. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment pathway analysis indicated these exosome lncRNAs mainly concerning response to chemokine-chemokine receptor interaction, which induced activated inflammation and structural remodeling. In addition, increased plasma levels of CXCR3 ligands, including CXCL4, CXCL9, CXCL10 and CXCL11, were accumulated in AF patient tissues. Conclusion Our study provides the transcriptome profile revealing pattern of circulating exosome lncRNAs in atrial structural remodeling, which bring valuable insights into improving prognosis and therapeutic targets for AF.


Patients in the study
The diagnosis of AF via the medical history, 12-lead electrocardiogram and 24 h electrocardiogram monitoring.The AF group contained paroxysmal and persistent AF patients, without permanent AF.We identified that paroxysmal AF returns to regular heartbeat within 7 days on its own or with treatment, and persistent AF last longer than seven days and cannot be able to regulate itself anymore.Patients attending cardiology department for non-AF-related conditions were screened and selected to create a sinus rhythm (SR) group.Some patients were excluded from SR group if they had any previous AF or other arrhythmia history.The patients of two groups were excluded if they had severe chronic kidney disease (eGFR < 30 mL/min), recent or active tumor, thyroid disease, acute myocardial infarction, stroke, other types of arrhythmias and acute heart failure.Finally, we selected 105 SR individuals and 110 AF patients enrolled in the current study.We also collected peripheral blood samples to obtain plasma via centrifuge (3500 rpm/min, 15 min, 4°C), then stored in-80°C freezer for further experiments.Besides, we harvested 15 atrial samples from patients accepting mitral valve replacement surgery, including 8 persistent AF patients and 7 individuals with SR.The atrial tissues were frozen immediately by liquid nitrogen during the surgery and then stored at-80°C.

Isolation, purification and identification of Exosomes
The methods of isolation, separation and purification in circulating exosomes were guided by recent protocols. [1]riefly, plasma samples were centrifuged at 300× g for 10 min, 2, 000× g for 30 min and 10, 000× g for 60 min.Then the supernatant was followed by filtration through a 0.22 μm filter to eliminate cellular debris.For further purification, the supernatant was ultracentrifuged at 120, 000× g for 90 min twice.The procedures of centrifugation were performed at 4°C.The exosome pellet was resuspended in 50 µL PBS.Then the morphology of exosome was observed by transmission electron microscope (EM, HT-7700, Hitachi, Japan).The expressions of exosomal specific surface markers including CD81 and Alix were measured via Western blot.

Exosome labeling
Exosomes were labeled with PKH26 Fluorescent Cell Linker Kit (Sigma-Aldrich, USA).Resuspended plasma exosomes were added to 150 µL of Diluent C (Sigma-Aldrich, USA).Then 2 µL of PKH26 dye was added to 500 µL of Diluent C and incubated with the exosome-Diluent C solution for 5 min at room temperature.The labeled exosomes were purified via centrifugation to remove the excess dye and resuspended in 100 µL of PBS for further experiments.Cardiomyocytes were stained by WGA.The uptake of labeled exosomes by cardiomyocytes was visualized by confocal microscopy (Zeiss, Germany).

Transthoracic Echocardiograph and Cardiac Magnetic Resonance
All patients had transthoracic echocardiography with tissue Doppler analysis performed by board certified physicians.LA diameter, RA diameter and LVEF were recorded.Cardiac magnetic resonance (CMR) detection was performed on a 3.0 T scanner according to standard protocols. [3]Left atrial appendage flow velocity (LAAFV) of AF individuals were measured by CMR.

Supplemental Figure 1 :
Profile of differential circulating exosome mRNAs in AF. (A) Heatmap of differential circulating exosome mRNAs between SR controls (n = 3) and AF patients (n = 3).(B) GO functional analysis of differential circulating exosomal mRNAs.(C) KEGG pathway analysis of differential circulating exosomal mRNAs.n represents the number of patients in every group.SR: sinus rhythm, AF: atrial fibrillation.Supplemental Figure 2: LAD and LAAFV of AF patients.(A) LAD of paroxysmal (n = 69) and persistent AF patients (n = 41) tested by Echocardiography.(B) LAAFV of paroxysmal (n = 69) and persistent AF patients (n = 41) tested by CMR.LAD: left atrial diameter, LAAFV: left atrial appendage flow velocity, CMR: cardiac magnetic resonance.n represents the number of patients in every group.Supplemental Figure 3: Negative correlation between several exosomal lncRNAs and atrial structure in AF patients.(A) Pearson analysis of circulating exosomal NR003045 correlated with LAD in AF patients, r = -0.1035and P = 0.2818.(B) Pearson analysis of circulating exosomal NONHSAT200958.1 correlated with LAD in AF patients, r = 0.2042 and P = 0.0323.(C) Pearson analysis of circulating exosomal NR003045 correlated with LAAFV in AF patients, r = 0.1144 and P = 0.2341.(D) Pearson analysis of circulating exosomal NONHSAT205820.1 correlated with LAAFV in AF patients, r = 0.1197 and P = 0.2131.LAD: left atrial diameter, LAAFV: left atrial appendage flow velocity.Supplemental Figure 4: Circulating exosomes from human plasma can be absorbed by atrial cardiomyocytes in vivo.(A) Scheme for human plasma exosomes preparation, transplant and AF induction in rats.(B) PKH26 and WGA staining in Exo-SR and Exo-AF rats, scale bar 50 μm (n = 3/group).(C) The lncRNA expressions in the atria of rats (n = 6/group).Supplemental Figure 5: Circulating exosomes from human plasma enhances AF susceptibility in rats.(A) Representative trace of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in Exo-SR and Exo-AF rats after programmed intracardiac stimulation (red line).(B) Incidence of pacinginduced AF in Exo-SR and Exo-AF rats (n = 6/group).(C) AF duration in Exo-SR and Exo-AF rats (n = 6/group).(D and E) NLRP3, Caspase 1 and CXCR3 protein levels in the atrium of rats (n = 6/group), Collagen 1 and α-SMA protein levels in the atrium of rats (n = 6/group).